The medical sciences are increasingly turning to purified mammalian antibodies as powerful diagnostic and therapeutic reagents. The ability to exploit the exquisite specificity of particular antibodies for their antigenic determinants has revolutionized the ways in which diseases are described, diagnosed and treated. For example, cancerous cells can be revealed by tagged antibodies directed against the cell-bound products of activated oncogenes. Cultured antibodies directed against unique epitopes on tumor surface antigens have been chemically coupled to cytotoxic agents and administered therapeutically.
Despite dramatic advances in in vitro antibody synthesis, such as the hybridoma method used to produce monoclonal antibodies, it is still relatively difficult to produce commercially acceptable quantities of purified antibody preparations. In particular, existing commercial methods can require large colonies of laboratory animals or large scale cell culture facilities, each of which comes at great expense. Moreover, purification of individual antibodies of interest from animals or from animal cell culture is laborious because many contaminating biomolecules must be removed from the preparation without destroying the conformational integrity and biological activity of the molecules.
As genes that encode desirable antibodies have become more readily available, efforts have shifted away from production in animals and animal cells and toward the goal of obtaining active animal-derived antibodies from plants and plant cell cultures. It is the hope of workers in this field to increase the yield while decreasing the unit cost of purified antibody preparations.
Hiatt and co-workers, using Agrobacterium-mediated gene transfer methods, have separately transferred expressible DNA constructs encoding immunoglobulin light chain genes and heavy chain genes into tobacco plants. After cross-pollinating the resultant transformed plants, F.sub.1 progeny plants were obtained, from which functional two-chain antibody molecules were isolated. The antibody accounted for about 1% of total extractable protein. Hiatt and others have also obtained antibody production in a tobacco cell culture after transferring by electroporation a single vector expressing both a heavy chain gene and a light chain gene.
PCT patent application WO 91/02066 discloses the transformation of tobacco suspension culture cells with a recombinant genetic construct encoding a single chain human serum albumin protein molecule fused to a N-terminal plant signal peptide. After transformation of the cells by electroporation, the human protein was detectable in the culture medium at a concentration of 2 ng per 10 microgram (0.02%) of extracellular protein.
Expression of antibody-like single-chain variable region fragment (sF.sub.v) proteins that bind antigens has been observed by Owen et al, Bio/Technology, 10:790 (1992) in transgenic tobacco plants at about 0.06-0.1% of total soluble protein.
The art has expressed frustration at an inability to achieve high yields of the desired antibodies. When the antibody of interest is produced at a low level, it is concomitantly harder to purify the antibody from the plant or plant extract.
DNA encoding sF.sub.v proteins has been cloned upstream from genes encoding a desired effector function, such as a cytotoxin, in place of the natural effector portion of an antibody molecule to create a novel protein fusion gene. This DNA has been expressed at low levels.